Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells.
Functional properties of the copper-transporting ATPase ATP7B (the Wilson's disease protein) expressed in insect cells.
复制标题
DOI：
10.1074/jbc.m109368200
复制
发表时间：
2002-01-11
影响因子：
4.8
通讯作者：
Lutsenko, S
中科院分区：
文献类型：
--
作者：
Tsivkovskii, R;Eisses, JF;Lutsenko, S
铜转运ATP酶ATP 7 B是铜在人体细胞中正常分布所必需的。ATP 7 B基因的突变导致铜在许多组织中积累，并导致严重的多系统疾病，称为威尔逊病。一级序列分析表明，铜转运ATP酶ATP 7 B或威尔逊病蛋白（WNDP）属于阳离子转运P型ATP酶大家族，但缺乏对其酶性质的详细表征。在这里，我们开发了一个杆状病毒介导的表达系统的WNDP，它允许直接和定量分析这种蛋白质的催化特性。使用这个系统，我们提供的实验证据表明，WNDP具有功能特性的P型ATP酶。它形成磷酸化中间体，对羟胺、碱性pH和ATP或ADP处理敏感。ATP以0.95 +/- 0.25 μ M的表观K-m刺激磷酸化; ADP以3.2 +/- 0.7 μ M的表观K-m促进去磷酸化。在保守序列基序DKTG中用Ala取代Asp（1027）可消除磷酸化作用，这与该残基在催化循环中作为磷酸受体的作用一致。WNDP的催化磷酸化被铜螯合剂浴铜灵抑制;铜以特定和合作的方式重新激活浴铜灵处理的WNDP，证实铜是形成酰基磷酸中间体所需的。这些研究建立了ATP 7 B铜转运ATP酶的关键催化特性，并为定量分析其在正常和病变细胞中的功能提供了基础。
Copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in the ATP7B gene lead to copper accumulation in a number of tissues and to a severe multisystem disorder, known as Wilson's disease. Primary sequence analysis suggests that the copper-transporting ATPase ATP7B or the Wilson's disease protein (WNDP) belongs to the large family of cation-transporting P-type ATPases, however, the detailed characterization of its enzymatic properties has been lacking. Here, we developed a baculovirus-mediated expression system for WNDP, which permits direct and quantitative analysis of catalytic properties of this protein. Using this system, we provide experimental evidence that WNDP has functional properties characteristic of a P-type ATPase. It forms a phosphorylated intermediate, which is sensitive to hydroxylamine, basic pH, and treatments with ATP or ADP. ATP stimulates phosphorylation with an apparent K-m of 0.95 +/- 0.25 muM; ADP promotes dephosphorylation with an apparent K-m of 3.2 +/- 0.7 mum. Replacement of Asp(1027) with Ala in a conserved sequence motif DKTG abolishes phosphorylation in agreement with the proposed role of this residue as an acceptor of phosphate during the catalytic cycle. Catalytic phosphorylation of WNDP is inhibited by the copper chelator bathocuproine; copper reactivates the bathocuproine-treated WNDP in a specific and cooperative fashion confirming that copper is required for formation of the acylphosphate intermediate. These studies establish the key catalytic properties of the ATP7B copper-transporting ATPase and provide a foundation for quantitative analysis of its function in normal and diseased cells.